This work addresses key issues in the field of membrane transport: (1) What is the overall organization of a transport protein? (2) What residues comprise the translocation pathway through the transporter? (3) What determines substrate specificity and selectivity? To answer these questions, we will combine biochemistry with site-directed and selection-driven mutagenesis of UhpT, a model transporter in Escherichia coli, We plan four kinds of experiments. A structural model of UhpT will be tested by placing pairs of cysteines in an otherwise cysteine-less UhpT. On exposure to oxidizing conditions, formation of disulfide bonds will serve as a molecular ruler to identify neighboring helices, revealing the overall organization of the transmembrane helical array. Concurrently we will develop an in vitro system, with purified and reconstituted protein to characterize residues that line the translocation pathway. We will probe single-cysteine variants with hydrophilic and impermeant thiol-reactive agents. Residues on the pathway, if replaced by cysteine, are accessible to such probes when they approach from either they extra- or intracellular face of the protein. Patterns of accessibility to probes differing in size, shape and charge will be used to learn about the way(s) in which substrates enter and leave the pathway. Current evidence implicates TM11 as a key player in setting substrate specificity in UhpT. To explore this question, we will select or engineer UhpT variants that have acquired novel features, including an altered substrate specificity and altered charge selectivity. A long term objective will be to obtain UhpT 2-dimensional crystals, so that structure-function relationships can be placed on a rational basis. Accordingly, we will establish conditions that yield milligram quantities of stable, purified UhpT for analysis by electron diffraction during trials of crystallography.